The present invention is directed to a device for improving the ability to detect components of a mixture in capillary electrochromatographic separations by eliminating band broadening at the transition region between a packed column and an analysis region.
In liquid chromatography, chemical separations can be performed by flowing a fluid (the mobile phase) past an immobilized material (the stationary phase) inside a liquid chromatography (LC) column. This technique is used for chemical analysis by injecting a sample consisting of multiple components into one end of the LC column, causing the individual components comprising the sample to be separated into distinct and discrete bands as the sample flows through the LC column, and detecting those bands near the exit end (typically within 2-3 channels diameters of the end of the stationary phase) of the LC column. Separation is governed by the dynamic partitioning of the analyte between the mobile phase and the stationary phase. Control of the separation may be achieved by adjusting the composition of the mobile phase or the stationary phase or both to influence analyte partitioning. In some chromatographic methods, such as capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) an electric field is applied to the chromatographic column to enhance separation.
In CEC, a capillary column is packed with a stationary phase material similar to that used in micro high pressure liquid chromatography (HPLC). The mobile phase, however, is caused to flow through the capillary column by an applied electric field that creates an electro-osmotic flow, similar to that in CZE, rather than using high pressure mechanical pumps, as in HPLC. The CEC approach can thus achieve the high efficiency of CZE. In addition, as in the case with micro-HPLC, CEC may be used to analyze neutral compounds that are not separable by CZE. The miniaturization of the separation column by using a capillary column in CEC offers several advantages, including improved efficiency, mass detection sensitivity, low solvent consumption, small sample quantity, and easier coupling to detector such as mass spectrometers and flame-based detectors.
A growing interest has developed in portable devices that will permit the rapid analysis of minute quantities of various chemical agents, and mixtures thereof, in a non-laboratory setting (i.e., xe2x80x9cin the fieldxe2x80x9d). Chromatography, and particularly CEC, offers a means for separating the various components of a mixture for subsequent detection and analysis xe2x80x9cin the fieldxe2x80x9d and numerous microcolumn chromatographic separations schemes have been developed that are capable of both rapid and efficient separation of complex mixtures.
However, these two goals of fast and efficient analysis of the components of a mixture tend to be mutually exclusive. Reduction in chromatographic analysis time is usually achieved by forcing the sample through the chromatographic column rapidly. This approach sacrifices much of the separation efficiency of the chromatographic column, resulting in a phenomenon known as band broadening which degrades the resolution of the separation, i.e., the ability to separate out compounds having similar chemical structures will be lost . A second approach can be to shorten the column length. However, here also there will be a decrease in column efficiency which can be partially offset by reducing the chromatographic dimensions, such as by reducing the particle size of the stationary phase. This miniaturization introduces a new set of problems and, even here, it has been observed that band broadening can take place.
As will be shown in greater detail below, it is possible to eliminate, or significantly reduce, broadening of eluted bands by modifications to the column packing and/or the ionic strength or pH of the solution. However, this solution can be quite complex, requiring significant trial and error. A simpler solution, contraction of the column diameter in the region immediately downstream the stationary phase, has been shown to be equally effective in eliminating broadening of bands being eluted from the stationary phase of the column.
By eliminating the phenomenon of band broadening in capillary electrochromatography (CEC) systems the present invention provides for a significant improvement in the resolution and thus, more accurate detection and analysis of the bands which comprise the components of the mixture being analyzed. As disclosed herein, the phenomenon of particular concern, band broadening, takes place in the transition region where bands corresponding to the components of a mixture being analyzed are eluted from the stationary phase and into an analysis section, a region typically within 2-3 column diameters of the stationary phase. A novel device for eliminating the phenomenon of band broadening comprising contraction of the capillary column diameter in the region immediately downstream the stationary phase is described. Contraction of the capillary column diameter is accomplished by joining an open capillary contiguous with the outlet end of a capillary column packed with a stationary phase, wherein the open column has a cross-sectional area of from about 0.2 to 0.5 times that of the packed capillary column.
It is, therefore an object of the present invention to provide a method for improving the resolution of non-pressure driven capillary chromatographic systems, and particularly for capillary electrochromatography (CEC) systems columns by minimizing or eliminating conditions which lead to spreading or broadening of eluted bands at the point of analysis.
A further object of this invention is to improve the sensitivity of CEC separations.